The determination of markers and marker indices is an integral component of modern medical diagnosis in most varied medical fields. Hence, for example, the determination of the relationship of high density lipoproteins (HDL) to low-density lipoproteins (LDL) is part of the routine for blood tests; the determination of the relationship of CD4 to CD8 positive blood cells is one of the most important parameters of AIDS diagnosis; in the diagnosis of tumors, for example breast cancer, the determination of the hormone receptor status is considered to be an indicator for treatment following an operation. Because most varied structures serve as markers, the methods of determination are also diverse: ELISA, RIA, chemical and/or biochemical titrations, FACS, luminometric, nephelometric, densitometric analyses, etc. which are well known to the skilled person.
In the diagnosis of tumors, thin tissue section examination and/or cytological examination of a sample is still the method of choice. For this purpose, the trained pathologist applies histochemical and immunohistochemical and/or cytochemical and immunocytochemical methods to an increasing degree. Thus, the hormone receptor status mentioned above is immunohistologically and/or immunocytologically determined in breast cancer. (Hereinafter, the term immunohistochemical encompasses immunohistological as well as immunocytological). In all marker determinations, the skilled person must ensure that the test systems are capable of being qualitatively and quantitatively validated. Validation of some markers may be done using calibration against an internal or external standard and the generation of calibration curves through parallel measurement against positive and negative controls. However, generally these validation methods are very expensive and time consuming.
In immunohistochemistry, such a validation, especially a quantitative validation, is not trivial, because commercial kits for the immunohistochemical marker portrayal either contain no internal control at all or, for example as with the hormone receptor, contain cell preparations that are either positive or negative, i.e. only permit a yes/no decision and therefore can only be seen in the best case as a semi quantitative validation.
In an immunocytochemical staining kit HercpTest™ Code No. K 5204 market by DAKO, a control slide for validating a staining run for breast cancer is known. This control slide comprises three discrete and not mixed groups of cell line cells with known intensity score. By controlling if the groups of cell line cells give the pre-known results in a staining process, the staining process can be validated.
Thus, in practice, the trained pathologist carries out parallel determination of the marker to be examined on well characterized and pre-tested cases from his archive. This method is, on the one hand, technically uncertain or at least controversial as a result of the known heterogeneity of tissues and, on the other hand, ethically questionable, which makes a commercial realization of this procedure impossible.
Since the beginning of the 20th century, parameters for the determination of the proliferation activity have already been an integral component in the histopathological diagnosis of tumors. Immunohistologically, the portrayal of structures that controls the cell division cycle is used as markers for proliferation. Many of the participating cell cycle-associated proteins are transiently expressed in single cell cycle phases. In contrast to this, the nuclear Ki-67 protein (Gerdes, J., et al., Int. J. Cancer, 1983, 31: 13-20) is detectable in all active phases of the cell cycle, i.e. G1, S, G2 and mitosis, whereas resting phase cells (GO) are consistently negative for this protein (Gerdes, J., et al., J. Immunol., 1984,133: 1710-1715). This means that the Ki-67 protein expression (Ki-67 marker index) can serve as a measure for the growth fraction of a given cell population. For this, antibodies against the Ki-67 protein have found broad use in histopathology, especially in numerous studies on the use as a marker for malignancy assessment in human neoplasias (Sawhney, N. and Hall, P. A., J. Pathol, 1992, 168:161-162; Schwarting, R., Lab. Invest., 1993, 68:597-599; Lelle, R. J., et al., Cancer, 1987, 59:83-88; Lokhorst, H. M., et al., J. Clin. Invest, 1987, 79:1401-1411; Gerdes, J. et al., Am. J. Path., 1987 128:330-334 and 129:486-492).
In retrospective studies on various tumor entities, the role of the Ki-67 marker index as a prognosis marker is discussed in a different manner. While an unanimous correlation between the survival time of patients and the Ki-67 marker index was described for malignant non-Hodgkin's lymphomas (Gerdes, J. et al., 1987, Lancet, ii 448-449; Grogan, T. M. et al., 1988 Blood, 71:1157-1160; Hall, P. A. et al., J. Pathol. 1988, 154:223-235), this has been controversially discussed in part for breast cancer: Harberg et al. (Prognostic and relevant therapy factors in breast cancer, Novartis Pharma Publishers, Nürnberg, 1997) found no correlation in multi-variate analysis, whereas Querzoli, P. et. al., J. Clin. Pathol. 1996 November; 49(11):926-930; Veronese, S. M. et al., Anticancer Res. 1995 November; 15(6B):2717-2722; Clahsen, P. C. et al., J. Clin Oncol. 1998 February; 16(2):470-479; Rozan, S. et al., Int. J. Cancer 1998 February; 79(1): 27-33; Jansen, R. L. et al., Br. J. Cancer 1998 August; 78(4):460-465; Molino, A. et al., Int. J. Cancer 1997 August; 74(4):433-437; conclusively prove that the portrayal of the Ki-67 protein in paraffin sections with the monoclonal antibody MIB-1 in the multi-variate statistic analysis is an independent prognostic parameter in breast cancer. Aaltoma et al., 1997, Eur. Urol. 32: 410-415 and Borre et al., 1998, J. Urol. 159: 1609-1614, demonstrated that the Ki-67 marker index is also an independent prognostic parameter for prostate carcinoma, whereas Coetze et al., 1997, J. Urol. 157: 214-218, questioned the value of the Ki-67 marker index as a prognostic indicator, and Uzoaru et al., 1998, J. Surg. Oncol. 67: 33-37, even concluded that the Ki-67 marker index does not permit any significant prognosis for the survival of patients.
As demonstrated above by means of example on the hormone receptors, a standardization of immunostaining techniques as well as an internal validation of the quantitative analysis have not been possible up to now even with the promotion of the Ki-67 marker index.